Project Summary Despite considerable research over the past 30 years, there is still no established effective treatment to improve recovery following spinal cord injury (SCI). In part, this reflects incomplete understanding of the complex secondary pathobiological mechanisms involved. The aim of our research is to understand the cellular and molecular mechanisms responsible for post-injury neuroinflammation in order to allow future development of novel therapies. The voltage-gated proton channel Hv1 is a newly discovered ion channel, highly expressed in resting microglia of the brain. Under pathological conditions, microglial Hv1 is required for NADPH oxidase (NOX)-dependent generation of ROS (reactive oxygen species) by providing charge compensation for exported electrons and relieving intracellular acidosis. Thus, Hv1 is a unique target for controlling multiple NOX activities and ROS production. However, neither the precise signaling mechanisms underlying this finding nor critical role of Hv1 in the pathophysiology of SCI are fully understood. Based on our preliminary data, we will test the hypothesis that microglial Hv1 functions as a key mechanism in neuroinflammation, through altered NOX2/ROS/IFN-? signaling that modulates microglia-astrocyte interaction, thus affecting long-term neurological outcomes after SCI. We will use systemic or microglial Hv1 KO, microglial NOX2 KO transgenic mice and in vivo and in vitro innovatively technologies to determine the mechanisms of SCI-triggered Hv1 elevation on post-injury neuroinflammation. Aim 1 will determine the function and mechanisms of the Hv1 in neuroinflammation after SCI. Multiple quantitative assessments of microglia-mediated neuroinflammation will be combined with genetic or pharmacological intervention targeting Hv1 to test the hypothesis that SCI-induced microglial Hv1 activation mediates detrimental neuroinflammation and functional deficits through altered microglial NOX2/ROS signaling. Aim 2 will elucidate the role of microglial NOX2 in post-injury neuroinflammation. We will utilize genetic intervention to delete Hv1-dependent up-regulation of NOX2 in microglia, and evaluate the effects on microglial NOX2 coupling to Hv1 on neuroinflammation after SCI. Aim 3 will identify critical role of Hv1/NOX2-derived ROS/IFN-? in SCI-chronic neuroinflammation through microglia-astrocyte interaction. Complimentary cellular, molecular, and genetic approaches will be used to test the hypothesis that Hv1/NOX2-mediated microglial ROS activates pro-inflammatory astrocytes resulting in secreting IFN? that in turn reinforces microglial inflammation, thus contributes to astrocytes dysfunction and neuronal damage. Our study will be the first to implicate microglial Hv1/NOX2/ROS/IFN-? signaling in the pathophysiology of SCI, leading to novel treatment approaches for SCI. Given the proposed roles for Hv1 in other inflammatory models, Hv1 signaling represents a generic mechanism relevant to other neuroinflammatory states.